Antisera were raised against γ-aminobutyric acid (GABA) or glutamate (Glu) conjugated to bovine serum albumin with glutaraldehyde. After purification, these antisera reacted strongly with fixed GABA or Glu, but not significantly with other amino acids fixed with glutaraldehyde to brain macromolecules. The antisera were used to demonstrate the distributions of Glu-like and GABA-like immunoreactivities (Glu-LI and GABA-LI) in parts of the perfusion-fixed mouse and rat brain, including the olfactory bulb, cerebral neocortex, thalamus, basal ganglia, lower brain stem, and cerebellum.
The level of GABA-LI varied widely among brain regions, thus it was very high in the globus pallidus and substantia nigra and low in the bulk of the thalamus. The GABA antisera labeled nonpyramidal neurons of the neocortex, most cells of the reticular nucleus of the thalamus, medium-sized cells of the caudatoputamen, and stellate, basket, Golgi, and Purkinje cells of the cerebellum. The distribution of GABA-LI closely matched that of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), as revealed in immunocytochemical studies by others. However, the GABA antisera seem to be better suited than GAD antisera for demonstrating putative GABA-ergic axons. The results suggest that GABA-LI, as displayed by the present method, is a good marker of neurons thought to use GABA as a transmitter.
Glutamate-like immunoreactivity was much more evenly distributed among regions than GABA-LI, but was particularly low in globus pallidus and substantia nigra and high in the cerebral cortex. Mitral cells of the olfactory bulb, pyramidal neocortical cells, and other cells assumed to use Glu or aspartate as transmitter were stained for Glu-LI, but so also were neurons that are thought to use other transmitters, such as cells in the substantia nigra pars compacta, in the dorsal raphe nucleus, and in the brain stem motor nuclei. The Glu antisera seem to reveal the “transmitter pool” as well as the “metabolic pool” of Glu in perfusion-fixed material.
This report shows that it is possible by means of immunocytochemistry to display reliably the tissue contents of GABA and Glu in material that has been fixed by perfusion with glutaraldehyde.